Extracellular serine protease cascades modulate protective responses to limit bleeding and infection in vertebrate and invertebrate animals, but our understanding of these pathways in insects is rudimentary at best. Previous work from this laboratory has established the lepidopteran insect, Manduca sexta, as a model system well suited for biochemical characterization of the complex functions of hemolymph proteases. Experiments in this proposal are designed to investigate the roles of plasma serine proteases and their inhibitors (serpins) in insect innate immune responses, including activation of phenoloxidase. The work will test hypotheses based on the following model. Pattern recognition proteins in plasma bind to microbial surfaces. These proteins then interact with serine protease zymogens and other protein cofactors to form protein complexes, which localize the protease cascade and subsequent melanin deposition to the foreign surface. Separate pathways (or separate branches of converging pathways) may be initiated by different types of microorganisms, as they are recognized by separate sets of pattern recognition proteins. Inhibition of the proteases by specific interactions with serpins limit the duration and location of the response. The specific aims of the projectare: 1. Investigate the activation of selected clip domain proteases and their inhibition by specific serpins, to determine their order in microbe-activated cascade pathways. 2. Identify protein interactions that result in formation of protease activation complexes and investigate the assembly of such complexes and their roles in localizing phenoloxidase activation on surfaces. Relevance: Innate immune responses are likely to affect the outcome of infections of insect vectors with the pathogens and parasites they transmit. The long term goals of the research are to gain a thorough understanding of the protease cascades that mediate innate immune responses in M. sexta, to apply this knowledge to insect vectors of human diseases, and to apply advantages of the M. sexta system for fundamental studies on the regulation of serine protease activity.